Selection of biological objects

Abstract

Provided herein are molecular automaton systems for identification, isolation, or elimination of a target biological object. Some embodiments include modules specific for a target biological object having a first biological object surface marker and a second biological object surface marker. Some embodiments include modules specific for a target biological object having a first biological object surface marker but not a second biological object surface marker.

The invention claimed is: 
     
       1. A molecular automaton system for isolation, elimination, or treatment of a target biological object,
 (I) where the target biological object comprises a first object surface marker and a second object surface marker, the system comprises
 (a) a first target marker comprising
 (i) a first target-specific agent specific for the first object surface marker, and 
 (ii) a first double strand complex comprising a first oligonucleotide and a second oligonucleotide, the second oligonucleotide linked to the first target-specific agent; 
 
 (b) a second target marker comprising
 (i) a second target-specific agent specific for the second object surface marker, and 
 (ii) a second double strand complex comprising a third oligonucleotide and a fourth oligonucleotide, the fourth oligonucleotide linked to the second target-specific agent; 
 
 (c) a single stranded fifth oligonucleotide; and 
 (d) a single stranded sixth oligonucleotide linked to an isolation agent, a cytotoxic agent, or a therapeutic agent; 
 
 wherein,
 the first oligonucleotide has more complementarity for the fifth oligonucleotide than for the second oligonucleotide, such that when in proximity, the fifth oligonucleotide will disrupt the first double strand complex to form a single stranded second oligonucleotide and a third double strand complex comprising the first oligonucleotide and the fifth oligonucleotide; 
 the third oligonucleotide has more complementarity for the second oligonucleotide than for the fourth oligonucleotide, such that when in proximity, the single stranded second oligonucleotide will disrupt the second double strand complex to form a single stranded fourth oligonucleotide and a fourth double strand complex comprising the second oligonucleotide and the third oligonucleotide, the fourth double strand complex linked to the first target-specific agent via the second oligonucleotide, and the single stranded fourth oligonucleotide linked to the second target-specific agent; and 
 the sixth oligonucleotide has sufficient complementarity to the single stranded fourth oligonucleotide to form a fifth double strand complex therewith, but has insufficient complementarity for the fourth oligonucleotide to disrupt the second double strand complex; or 
 
 (II) where the target biological object comprises a first object surface marker but not a second object surface marker, the system comprises
 (a) a first target marker comprising
 (i) a first target-specific agent specific for the first object surface marker, and 
 (ii) a first double strand complex comprising a first oligonucleotide and a second oligonucleotide, the second oligonucleotide linked to the first target-specific agent; 
 
 (b) a second target marker comprising
 (i) a second target-specific agent specific for the second object surface marker, and 
 (ii) a second double strand complex comprising a third oligonucleotide and a fourth oligonucleotide, the fourth oligonucleotide linked to the second target-specific agent; 
 
 (c) a single stranded fifth oligonucleotide; 
 (d) a sixth double strand complex comprising a sixth oligonucleotide and a seventh oligonucleotide, the sixth oligonucleotide linked to an isolation agent, a cytotoxic agent, or a therapeutic agent; 
 
 wherein,
 the first oligonucleotide has more complementarity for the fifth oligonucleotide than for the second oligonucleotide, such that when in proximity, the fifth oligonucleotide will disrupt the first double strand complex to form a single stranded second oligonucleotide and a third double strand complex comprising the first oligonucleotide and the fifth oligonucleotide; 
 the third oligonucleotide has more complementarity for the second oligonucleotide than for the fourth oligonucleotide, such that when in proximity, the single stranded second oligonucleotide will disrupt the second double strand complex to form a single stranded fourth oligonucleotide and a fourth double strand complex comprising the second oligonucleotide and the third oligonucleotide, the fourth double strand complex linked to the first target-specific agent via the second oligonucleotide, and the single stranded fourth oligonucleotide linked to the second target-specific agent; 
 the sixth oligonucleotide has more complementarity for the second oligonucleotide than for the seventh oligonucleotide, such that when in proximity, the single stranded second oligonucleotide will disrupt the sixth double strand complex to form a single stranded seventh oligonucleotide and a seventh double strand complex comprising the second oligonucleotide and the sixth oligonucleotide, the seventh double strand complex linked to the first target-specific agent via the second oligonucleotide, and the single stranded fourth oligonucleotide linked to the second target-specific agent; and 
 the third oligonucleotide has more complementarity for the second oligonucleotide than the sixth oligonucleotide has for the second oligonucleotide, such that when in proximity, the sixth oligonucleotide cannot displace the third oligonucleotide from the fourth double strand complex comprising the second oligonucleotide and the third oligonucleotide. 
 
 
     
     
       2. The system of  claim 1 , wherein the system forms a marked target biological object. 
     
     
       3. The system of  claim 1 , wherein the target biological object comprises at least one of a cell, an organelle, or a vesicle. 
     
     
       4. The system of  claim 1 , wherein the target biological object comprises at least one of a stem cell, a leukocyte group, a granulocytes, a monocyte, a T lymphocyte, a T helper cell, a T regulatory cell, a cytotoxic T cell, a naïve T cell, a lymphocyte, a thrombocyte, or a natural killer cell. 
     
     
       5. The system of  claim 4 , wherein the target biological object comprises at least one of a natural killer cell, a T-cell, or a B-cell. 
     
     
       6. The system of  claim 1 , wherein the target biological object is selected from the group consisting of an exosome, apoptotic bleb, shedding vesicle, microparticle, prostasome, tolerosome, prom inosome, unilamellar liposome vesicle, or multilamellar liposome vesicle, vacuole, plant vacuole, contractile vacuole, lysosome, peroxisome, transport vesicle, secretory vesicle, synaptic vesicle, hormonal secretory vesicle, cell wall-associated vesicle, toxic membrane vesicle, signal molecule vesicle, gas vesicle, membrane vesicle, matrix vesicle, multivesicular body, outer membrane vesicle, mitochondria, plastic, flagellum, endoplasmic reticulum, Golgi apparatus, vacuole, nucleus, acrosome, autophagosome, centriole, cilium, eyespot apparatus, glycosome, glyoxosome, hydrogenosome, lysosome, melanosome, mitosome, myofibril, nematocyst, nucleolus, parenthesome, peroxisome, proteasome, ribosome, 80s ribosome, vesicle, nucleosome, microtubule, large RNA and RNA-protein complex, ribosome, spliceosome, vault, proteasome, DNA polymerase III holoenzyme, RNA polymerase II holoenzyme, symmetric viral capsid, complex of GroEL and GroES, membrane protein complex, photosystem I, ATP synthase, large DNA and DNA-protein complex, nucleosome, centriole and microtubule-organizing center, cytoskeleton, nucleolus, carboxysome, chlorosome, magnetosome, nucleoid, plasmid, ribosome, 70s ribosome, thylakoid, and mesasome. 
     
     
       7. The system of  claim 1 , wherein the target biological object is produced by or associated with at least one of a stem cell, a leukocyte group, a granulocytes, a monocyte, a T lymphocyte, a T helper cell, a T regulatory cell, a cytotoxic T cell, a naïve T cell, a lymphocyte, a thrombocyte, or a natural killer cell. 
     
     
       8. The system of  claim 7 , wherein the target biological object is produced by or associated with at least one of a natural killer cell, a T-cell, or a B-cell. 
     
     
       9. The system of  claim 1 , wherein
 the target biological object is a stem cell and the first object surface marker or the second object surface marker is selected from the group consisting of CD34+, CD31−, and CD117; 
 the target biological object is a leukocyte group and the first object urface marker or the second object surface marker is CD45+; 
 the target biological object is a granulocyte and the first object surface marker or the second object surface marker is selected from the group consisting of CD45+, CD11 b, CD15+, CD24+, CD114+, and CD182+; 
 the target biological object is a monocyte and the first object surface marker or the second object surface marker is selected from the group consisting of CD45+, CD14+, CD114+, CD11a, CD11 b, CD91+, CD16+; 
 the target biological object is a T lymphocyte and the first object surface marker or the second object surface marker is selected from the group consisting of CD45+ and CD3+; 
 the target biological object is a T helper cell and the first object surface marker or the second object surface marker is selected from the group consisting of CD45+, CD3+, and CD4+; 
 the target biological object is a T regulatory cell and the first object surface marker or the second object surface marker is selected from the group consisting of CD4, CD25, and Foxp3; 
 the target biological object is a Cytotoxic T cell and the first object surface marker or the second object surface marker is selected from the group consisting of CD45+, CD3+, and CD8+; 
 the target biological object is a naïve T-cell and the first object surface marker or the second object surface marker is selected from the group consisting of CD45RA+ and CD3+; 
 the target biological object is a B lymphocyte and the first object surface marker or the second object surface marker is selected from the group consisting of CD45+, CD19+ or CD45+, CD20+, CD24+, CD38, and CD22; 
 the target biological object is a Thrombocyte and the first object surface marker or the second object surface marker is selected from the group consisting of CD45+ and CD61+; or 
 the target biological object is a Natural killer cell and the first object surface marker or the second object surface marker is selected from the group consisting of CD16+, CD56+, CD3−, CD31, CD30, and CD38. 
 
     
     
       10. The system of  claim 1 , wherein:
 the first target-specific agent comprises a first antibody specific for the first object surface marker; and 
 the second target-specific agent comprises a second antibody specific for the second object surface marker. 
 
     
     
       11. The system of  claim 10 , wherein:
 the first target-specific agent comprises a first monoclonal antibody specific for the first object surface marker; and 
 the second target-specific agent comprises a second monoclonal antibody specific for the second object surface marker. 
 
     
     
       12. The system of  claim 1 , wherein the first object surface marker or the second object surface marker is selected from the group consisting of a Type 1 receptor, Type 2 G protein-coupled receptor, Type 3 kinase linked receptor, and Type 4 nuclear receptor. 
     
     
       13. The system of  claim 1 , wherein the first object surface marker or the second object surface marker is selected from the group consisting of an immune receptor, pattern recognition receptor, Toll-like receptor, killer activated receptor and killer inhibitor receptor, complement receptor, Fc receptor, B cell receptor, T cell receptor, cytokine receptor, ion channel linked receptor, nicotinic acetylcholine receptor, glycine receptor, gamma-aminobutyric acid receptor, gamma-aminobutyric acid A receptor, gamma-aminobutyric acid C receptor, glutamate receptor, N-methyl-D-aspartate receptor, α amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, Kainate receptor, 5-HT3 receptor, P2X receptor, cyclic nucleotide-gated ion channel, Inositol triphosphate receptor, intracellular ATP receptor, and ryanodine receptor. 
     
     
       14. The system of  claim 1 , wherein the first object surface marker or the second object surface marker is selected from the group consisting of a clathrin coat-associated marker, COPI coat-associated marker, COPII coat-associated marker, coatomer coat-associated marker, SNARE marker, v-SNARE, t-SNARE, Qa SNARE, Qb SNARE, Qc SNARE, and R SNARE. 
     
     
       15. The system of  claim 1 , wherein the first object surface marker or the second object surface marker is a small molecule selected from the group consisting of a steroid or nitro-phenol compound. 
     
     
       16. The system of  claim 1 , wherein the first oligonucleotide, the second oligonucleotide, the third oligonucleotide, the fourth oligonucleotide, the fifth oligonucleotide, the sixth oligonucleotide, or the seventh oligonucleotide comprise about 10 to about 100 nucleotides. 
     
     
       17. The system of  claim 16 , wherein the first oligonucleotide, the second oligonucleotide, the third oligonucleotide, the fourth oligonucleotide, the fifth oligonucleotide, the sixth oligonucleotide, or the seventh oligonucleotide comprise about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 nucleotides. 
     
     
       18. The system of  claim 1 , wherein more complementarity comprises about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%. 
     
     
       19. The system of  claim 1 , wherein
 a double strand complex comprises a pair of oligonucleotides having a difference in nucleotide number selected from the group consisting of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, and about 25 nucleotides; and 
 the difference in nucleotide number creates a toe hold sufficient to drive a strand-displacement reaction. 
 
     
     
       20. The system of  claim 1 , wherein the target biological object is isolated according to at least one of flow cytometry, fluorescence-activated cell sorting, magnetic-activated cell sorting, Cytometric Bead Array, a magnetic nanoparticle coated with an anti-fluorochrome antibody, superparamagnetic spherical polymer particles, polymer beads coated with an anti-fluorochrome antibody, avidin, or streptavidin, or plasmapheresis. 
     
     
       21. A method for isolating, eliminating, or treating a target biological object with the molecular automaton system of  claim 1 , comprising:
 (I) (a) contacting the first target marker, the second target marker, and a population of biological objects optionally comprising the target biological object, the target biological object comprising the first object surface marker and the second object surface marker, to form a marked target biological object; and 
 (b) contacting the single stranded fifth oligonucleotide and the single stranded sixth oligonucleotide linked to the isolation agent, the cytotoxic agent, or the therapeutic agent with the marked target biological object; or 
 (II) (a) contacting the first target marker, the second target marker, and a population of biological objects optionally comprising the target biological object, the target biological object comprising the first object surface marker but not the second object surface marker, to form a marked target biological object; and 
 (b) contacting the single stranded fifth oligonucleotide and the sixth double strand complex linked to the isolation agent, the cytotoxic agent, or the therapeutic agent with the marked target biological object. 
 
     
     
       22. The method of  claim 21 , wherein the target biological object comprises a cell, an organelle, or a vesicle. 
     
     
       23. The method of  claim 21 , wherein the target biological object comprises a stem cell, a leukocyte group, a granulocytes, a monocyte, a T lymphocyte, a T helper cell, a T regulatory cell, a cytotoxic T cell, a nave T cell, a lymphocyte, a thrombocyte, or a natural killer cell. 
     
     
       24. The method of  claim 23 , wherein the target biological object comprises at least one of a natural killer cell, a T-cell, or a B-cell. 
     
     
       25. The method of  claim 21 , wherein the target biological object is selected from the group consisting of an exosome, apoptotic bleb, shedding vesicle, microparticle, prostasome, tolerosome, prominosome, unilamellar liposome vesicle, or multilamellar liposome vesicle, vacuole, plant vacuole, contractile vacuole, lysosome, peroxisome, transport vesicle, secretory vesicle, synaptic vesicle, hormonal secretory vesicle, cell wall-associated vesicle, toxic membrane vesicle, signal molecule vesicle, gas vesicle, membrane vesicle, matrix vesicle, multivesicular body, outer membrane vesicle, mitochondria, plastic, flagellum, endoplasmic reticulum, Golgi apparatus, vacuole, nucleus, acrosome, autophagosome, centriole, cilium, eyespot apparatus, glycosome, glyoxosome, hydrogenosome, lysosome, melanosome, mitosome, myofibril, nematocyst, nucleolus, parenthesome, peroxisome, proteasome, ribosome, 80s ribosome, vesicle, nucleosome, microtubule, large RNA and RNA-protein complex, ribosome, spliceosome, vault, proteasome, DNA polymerase III holoenzyme, RNA polymerase II holoenzyme, symmetric viral capsid, complex of GroEL and GroES, membrane protein complex, photosystem I, ATP synthase, large DNA and DNA-protein complex, nucleosome, centriole and microtubule-organizing center, cytoskeleton, nucleolus, carboxysome, chlorosome, magnetosome, nucleoid, plasmid, ribosome, 70s ribosome, thylakoid, and mesasome. 
     
     
       26. The method of  claim 21 , wherein the target biological object is produced by or associated with a stem cell, a leukocyte group, a granulocytes, a monocyte, a T lymphocyte, a T helper cell, a T regulatory cell, a cytotoxic T cell, a nave T cell, a lymphocyte, a thrombocyte, or a natural killer cell. 
     
     
       27. The method of  claim 26 , wherein the target biological object is produced by or associated with at least one of a natural killer cell, a T-cell, or a B-cell. 
     
     
       28. The method of  claim 21 , wherein
 the target biological object is a stem cell and the first object surface marker or the second object surface marker is selected from the group consisting of CD34+, CD31−, and CD117; 
 the target biological object is a leukocyte group and the first object surface marker or the second object surface marker is CD45+; 
 the target biological object is a granulocyte and the first object surface marker or the second object surface marker is selected from the group consisting of CD45+, CD11 b, CD15+, CD24+, CD114+, and CD182+; 
 the target biological object is a monocyte and the first object surface marker or the second object surface marker is selected from the group consisting of CD45+, CD14+, CD114+, CD11a, CD11 b, CD91+, CD16+; 
 the target biological object is a T lymphocyte and the first object surface marker or the second object surface marker is selected from the group consisting of CD45+ and CD3+; 
 the target biological object is a T helper cell and the first object surface marker or the second object surface marker is selected from the group consisting of CD45+, CD3+, and CD4+; 
 the target biological object is a T regulatory cell and the first object surface marker or the second object surface marker is selected from the group consisting of CD4, CD25, and Foxp3; 
 the target biological object is a Cytotoxic T cell and the first object surface marker or the second object surface marker is selected from the group consisting of CD45+, CD3+, and CD8+; 
 the target biological object is a naïve T-cell and the first object surface marker or the second object surface marker is selected from the group consisting of CD45RA+ and CD3+; 
 the target biological object is a B lymphocyte and the first object surface marker or the second object surface marker is selected from the group consisting of CD45+, CD19+ or CD45+, CD20+, CD24+, CD38, and CD22; 
 the target biological object is a Thrombocyte and the first object surface marker or the second object surface marker is selected from the group consisting of CD45+ and CD61+; or 
 the target biological object is a Natural killer cell and the first object surface marker or the second object surface marker is selected from the group consisting of CD16+, CD56+, CD3-, CD31, CD30, and CD38. 
 
     
     
       29. The method of  claim 21 , wherein:
 the first target-specific agent comprises a first antibody specific for the first object surface marker; and 
 the second target-specific agent comprises a second antibody specific for the second object surface marker. 
 
     
     
       30. The method of  claim 21 , wherein:
 the first target-specific agent comprises a first monoclonal antibody specific for the first object surface marker; and 
 the second target-specific agent comprises a second monoclonal antibody specific for the second object surface marker. 
 
     
     
       31. The method of  claim 21 , wherein the first object surface marker or the second object surface marker is selected from the group consisting of a Type 1 receptor, Type 2 G protein-coupled receptor, Type 3 kinase linked receptor, and Type 4 nuclear receptor. 
     
     
       32. The method of  claim 21 , wherein the first object surface marker or the second object surface marker is selected from the group consisting of an immune receptor, pattern recognition receptor, Toll-like receptor, killer activated receptor and killer inhibitor receptor, complement receptor, Fc receptor, B cell receptor, T cell receptor, cytokine receptor, ion channel linked receptor, nicotinic acetylcholine receptor, glycine receptor, GABA gamma-aminobutyric acid receptor, gamma-aminobutyric acid A receptor, gamma-aminobutyric acid C receptor, glutamate receptor, methyl-D-aspartate receptor, α amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, Kainate receptor, 5-HT3 receptor, P2X receptor, cyclic nucleotide-gated ion channel, Inositol triphosphate receptor, intracellular ATP receptor, and ryanodine receptor. 
     
     
       33. The method of  claim 21 , wherein the first object surface marker or the second object surface marker is selected from the group consisting of a clathrin coat-associated marker, COPI coat-associated marker, COPII coat-associated marker, coatomer coat-associated marker, SNARE marker, v-SNARE, t-SNARE, Qa SNARE, Qb SNARE, Qc SNARE, and R SNARE. 
     
     
       34. The method of  claim 21 , wherein the first object surface marker or the second object surface marker is a small molecule selected from the group consisting of a steroid or nitro-phenol compound. 
     
     
       35. The method of  claim 21 , wherein the first oligonucleotide, the second oligonucleotide, the third oligonucleotide, the fourth oligonucleotide, the fifth oligonucleotide, the sixth oligonucleotide, or the seventh oligonucleotide comprise about 10 to about 100 nucleotides. 
     
     
       36. The method of  claim 29 , wherein the first oligonucleotide, the second oligonucleotide, the third oligonucleotide, the fourth oligonucleotide, the fifth oligonucleotide, the sixth oligonucleotide, or the seventh oligonucleotide comprise about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 nucleotides. 
     
     
       37. The method of  claim 21 , wherein more complementarity comprises about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%. 
     
     
       38. The method of  claim 21 , wherein
 a double strand complex comprises a pair of oligonucleotides having a difference in nucleotide number selected from the group consisting of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, and about 25 nucleotides; and 
 the difference in nucleotide number creates a toe hold sufficient to drive a strand-displacement reaction. 
 
     
     
       39. The method of  claim 2 , wherein the target biological object is isolated according to at least one of flow cytometry, fluorescence-activated cell sorting, magnetic-activated cell sorting, Cytometric Bead Array, a magnetic nanoparticle coated with an anti-fluorochrome antibody, superparamagnetic spherical polymer particles, polymer beads coated with an anti-fluorochrome antibody, avidin, or streptavidin, or plasmapheresis.